circPLIN2 promotes clear cell renal cell carcinoma progression by binding IGF2BP proteins and miR-199a-3p

Recent evidence has indicated that circular RNAs (circRNAs), a novel type of regulatory RNA, play important roles in the development and progression of various cancers. However, the potential regulatory roles and molecular mechanisms of circRNAs in clear cell renal cell carcinoma (ccRCC) remain largely unclear. Here, we explored circRNA expression profiles in 10 paired samples of RCC (including cancer tissues and surrounding tissues) from the Gene Expression Omnibus (GEO) datasets GSE124453 and GSE108735. We initially identified hsa_circ_0086457, designated circPLIN2, derived from exons 4 to 5 of the PLIN2 gene. We observed that circPLIN2 was preferentially located in the cytoplasm and was more stable than its linear counterpart PLIN2. circPLIN2 was significantly upregulated in ccRCC cells and tissues, and its overexpression was correlated with higher clinical stage and worse prognosis for ccRCC patients. Moreover, gain- and loss-of-function assays indicated that circPLIN2 promoted ccRCC cell proliferation, migration, and invasion in vitro and ccRCC tumor growth and metastasis in vivo. Mechanistically, circPLIN2 not only increased the stability of the c-Myc and MARCKSL1 mRNAs by binding to the KH domains of IGF2BP proteins but also competitively sponged miR-199a-3p to abolish the repressive effect of miR-199a-3p on ZEB1 expression, which ultimately resulted in ccRCC tumorigenesis and progression. Collectively, our results suggest that circPLIN2 may represent a promising diagnostic and prognostic biomarker and a potential therapeutic target for ccRCC patients.


Introduction
Renal cell carcinoma (RCC) is one of the most common malignant tumors in humans, and its high morbidity and mortality, with 73,750 new cases and 14,830 deaths estimated for 2020 in the US, make it a growing global health problem [1]. Clear cell renal cell carcinoma (ccRCC) is the most common type of RCC, accounting for approximately 70-75% of RCC types [2]. Currently, the gold standard for the diagnosis and treatment of ccRCC patients is the early detection of microtumor lesions and radical surgical resection of localized ccRCC, which generally result in excellent long-term disease-free survival (DFS) [3,4]. However, the prognosis for patients with advanced ccRCC is poor due to local tumor recurrence or distant metastasis even after radical nephrectomy [5]. Additionally, the majority of ccRCCs are resistant to both traditional chemotherapy and radiotherapy once they recur or metastasize, which leads to lower overall survival for advanced ccRCC patients [6,7]. Therefore, it is urgent to elucidate the potential mechanisms in the pathogenesis of ccRCC and identify new effective therapeutic approaches for ccRCC.
Recently, circular RNAs (circRNAs) have been characterized by covalently closed loop structures, without a 5' cap and a 3' poly(A) tail, which are formed by back splicing events and have attracted the attention of many researchers [8][9][10]. circRNAs are widely involved in a variety of eukaryotes and have more stability and a stronger resistance to digestion by RNase R treatment than their linear counterpart mRNAs [8][9][10].
In this study, we investigated the expression pro les of circRNAs in 10 paired samples of RCC (including cancer tissues and surrounding tissues) from the GEO datasets GSE124453 and GSE108735. We initially identi ed hsa_circ_0086457, termed circPLIN2, derived from exons 4 to 5 of the PLIN2 gene. circPLIN2 was markedly upregulated in ccRCC cells and tissues, and its overexpression was correlated with higher clinical stage and worse prognosis in ccRCC patients. circPLIN2 was preferentially located in the cytoplasm and had more stability than its linear transcript PLIN2. Gain-and loss-of-function assays indicated that elevated circPLIN2 promoted ccRCC cell proliferation, migration and invasion in vitro and ccRCC tumor growth in vivo. Mechanistically, circPLIN2 not only enhanced the mRNA stability of c-Myc and MARCKSL1 by binding to the KH domains of IGF2BP proteins but also competitively sponged miR-199a-3p to abolish the repressive effect of miR-199a-3p on ZEB1, which ultimately resulted in tumorigenesis and progression of ccRCC. Results circPLIN2 is signi cantly upregulated in ccRCC cells and tissues and participates in the progression of ccRCC To explore the regulatory roles of circRNAs and their underlying molecular mechanisms in the development and progression of human ccRCC, we rst analyzed the expression pro les of circRNAs in human ccRCC. We performed a joint analysis of the circRNA expression data for a total of 10 paired samples of RCC (including cancer tissues and surrounding tissues) from the GEO datasets GSE124453 and GSE108735 (http://www.ncbi.nlm.nih.gov/geo) ( Fig. 1A 1C). In situ hybridization staining was performed on a tissue microarray of human ccRCC, including 90 cases of tumor tissues and adjacent tissues, with probes speci c for circPLIN2 to validate its expression. Three representative cases of in situ hybridization staining for circPLIN2 expression in the tissue microarray were shown (Fig. 1D). We found that circPLIN2 was signi cantly upregulated in ccRCC tissues compared with surrounding normal tissues (Fig. 1E left), accounting for approximately 63% (57/90) of 90 ccRCC specimens (Fig. 1E right). To further examine circPLIN2 overexpression in ccRCC, we used a panel of four human ccRCC cell lines (786-O, ACHN, 769-P and OS-RC-2) and HK-2 cells (a proximal tubule epithelial cell line) to test circPLIN2 expression by RT-qPCR. The results showed that circPLIN2 was observably overexpressed in ccRCC cells compared to HK-2 cells (Fig. 1F), which was consistent with the results of in situ hybridization staining assays ( Fig. 1D-E).
Further analysis indicated that circPLIN2 levels were dramatically higher in ccRCC tissues at the advanced American Joint Committee on Cancer (AJCC) stages (AJCC 3-4 stages) than in ccRCC tissues at the AJCC early stages (AJCC 1-2 stages) (Fig. 1G). Additionally, we analyzed the correlation between circPLIN2 expression and clinicopathological characteristics in 90 ccRCC patients. The results showed that circPLIN2 expression was only signi cantly correlated with tumor differentiation, and the higher the expression level of circPLIN2 was, the worse the tumor differentiation and the higher the malignant grade of the tumor ( Table 1). The survival curve analysis showed that ccRCC patients with high circPLIN2 expression had a markedly lower overall survival rate than ccRCC patients with low circPLIN2 expression (Fig. 1H). Moreover, the univariate Cox proportional hazard regression analysis showed that the differential expression of circPLIN2 was signi cantly correlated with overall survival in 78 ccRCC patients (P=0.026) ( Table 2), which was consistent with the results of the Kaplan-Meier analysis (Fig. 1H). However, the multivariate Cox proportional hazard regression analysis showed that the differential expression of circPLIN2 was not associated with overall survival in 78 ccRCC patients (P=0.206) ( Table  2), which may be explained by the fact that the number of patients involved in the study was small or there were some factors that interfered with the true results. The receiver operating characteristic curve (ROC) results indicated that the expression level of circPLIN2 showed excellent diagnostic performance for cancer and paracancer (Fig. 1I), AJCC 1-2 stages and 3-4 stages (Fig. 1J), and survival and death of ccRCC patients (Fig. 1K). Collectively, these results suggested that circPLIN2 was signi cantly upregulated in ccRCC cells and tissues and that its overexpression was correlated with higher clinical stage and worse prognosis in ccRCC patients.
General characteristics of circPLIN2 circPLIN2 is a circular RNA molecule derived from exons 4 to 5 of the PLIN2 gene on human chromosome 9 (9p22.1) with a length of 369 nucleotides ( Fig. 2A). The back-splice junction of circPLIN2 was ampli ed using divergent primers and con rmed by Sanger sequencing, and the result was consistent with the circBase database annotation (http://www.circbase.org) ( Fig. 2A). Subsequently, PCR ampli cation assays and agarose gel electrophoresis assays using divergent and convergent primers further demonstrated that circPLIN2 and its linear isoform PLIN2 both truly existed in ccRCC cells (Fig. 2B). We next investigated the resistance of circPLIN2 to digestion by RNase R treatment, and the results indicated that circPLIN2 was more tolerant to RNase R digestion than the linear counterpart PLIN2 (Fig. 2C). In addition, actinomycin D, an inhibitor of transcription, was used to test the half-life of circPLIN2 in ccRCC cells, and the results showed that the content of circPLIN2 decreased slowly over time compared with the linear transcript PLIN2 in 786-O cells in the presence of 2 μg/mL actinomycin D, suggesting that circPLIN2 had more stability or a longer half-life than its linear counterpart PLIN2 (Fig. 2D). To explore the cellular localization of circPLIN2, we performed RT-qPCR analysis to determine the abundance of nuclear and cytoplasmic circPLIN2 in ccRCC cells. The results showed that circPLIN2 was preferentially located in the cytoplasm of ACHN (Fig. 2E) and OS-RC-2 ( Fig. 2F) cells, which was consistent with the results of the uorescence in situ hybridization (FISH) assays ( Fig. 2G-H). Overall, circPLIN2, the back-spliced product of the parent gene PLIN2, was preferentially distributed in the cytoplasm of ccRCC cells and had a longer half-life and a stronger resistance to RNase R digestion than its linear counterpart PLIN2.

circPLIN2 promotes the proliferation, migration and invasion of ccRCC cells in vitro
To investigate whether changes in the expression of circPLIN2 affected the biological behaviors of ccRCC cells, two small interfering RNAs (circPLIN2-siRNA 1 and circPLIN2-siRNA 2) were designed and synthesized speci cally targeting the back-splice junction of circPLIN2, and a circPLIN2 overexpression vector was designed and constructed. The results of RT-qPCR assays showed that these two siRNAs could speci cally knock down the expression level of circPLIN2 in ACHN and OS-RC-2 cells but had no effect on PLIN2 mRNA expression (Fig. 3A). Similarly, circPLIN2 was successfully overexpressed in ACHN and OS-RC-2 cells, while PLIN2 mRNA expression showed no obvious change ( CircRNAs have been shown to interact with RNA-binding proteins to regulate protein functions [15][16][17][18][19][20]. As circPLIN2 was mainly distributed in the cytoplasm, RNA pull-down assays (Tagged RNA a nity puri cation assays) were performed to explore the proteins bound to circPLIN2 in the cytoplasm, and then puri ed proteins were subjected to liquid chromatography-mass spectrometry (LC-MS) and western blot analyses (Fig. 4A). To make the results of LC-MS more reliable, we repeated the LC-MS analysis and used a stricter screening standard (unique peptide≥2) to analyze the results of the two LC-MS experiments, and 12 proteins bound to circPLIN2 were screened (Fig. 4B-C and Supplementary Table  3). In addition, puri ed proteins from RNA pull-down assays were subjected to SDS-PAGE gel silver staining analysis, and the results were shown as follows (Fig. 4D). Notably, there were three speci c silver-stained bands at approximately 70 kD appearing in the MS2-circPLIN2 lane compared to the control MS2-Vector lane (Fig. 4D). Combined with the molecular weight and intracellular localization of the proteins (Table 3), we speculated that these three speci c silver-stained bands may be the IGF2BP1, IGF2BP2 and IGF2BP3 proteins. Accordingly, we performed western blot assays for the puri ed proteins obtained from RNA pull-down assays to detect the IGF2BP1, IGF2BP2 and IGF2BP3 proteins. The results indicated that circPLIN2 interacted with IGF2BP proteins (Fig. 4E and Supplementary Fig. 1A), which was consistent with the results of the RNA immunoprecipitation assays (Fig. 4F).
Intriguingly, IGF2BP proteins can enhance the mRNA stability of downstream genes [35]. Therefore, we considered whether the interaction between circPLIN2 and IGF2BP proteins would affect the mRNA stability of downstream genes of IGF2BP proteins, such as FSCN1, TK1, c-Myc and MARCKSL1. RT-qPCR assays showed that knockdown of circPLIN2 signi cantly reduced the mRNA levels of c-Myc and MARCKSL1 but had no effect on the mRNA levels of FSCN1 and TK1 ( To further investigate the speci c circPLIN2-binding domains of IGF2BP proteins, we constructed GFPtagged wild-type and truncated IGF2BP plasmids (Fig. 4M). IGF2BP proteins have six key domains, including RRM1-2 domains and KH1-4 domains, and RNA immunoprecipitation assays showed that the enrichment of circPLIN2 was signi cantly reduced following removal of the KH1-4 domains of the IGF2BP proteins, indicating that the KH1-4 domains were required for circPLIN2 to directly bind to the IGF2BP proteins ( Fig. 4N-P). Interestingly, the KH domains were the key domains for the binding of IGF2BP proteins and c-Myc or MARCKSL1 mRNA [35], suggesting that the KH domains may provide a common place for action among circPLIN2, IGF2BP proteins and c-Myc or MARCKSL1 mRNA.
Collectively, these data suggested that circPLIN2 enhanced the mRNA stability of c-Myc and MARCKSL1 by binding to the KH domains of IGF2BP proteins.

Overexpression of c-Myc or MARCKSL1 alleviates the inhibition of circPLIN2 knockdown on the proliferation, migration and invasion of ccRCC cells in vitro
To explore whether c-Myc and MARCKSL1 are involved in the circPLIN2-regulated development and progression of ccRCC, we constructed overexpression vectors of c-Myc and MARCKSL1. CCK-8 cell viability assays showed that knockdown of circPLIN2 signi cantly inhibited the proliferation of ACHN and OS-RC-2 cells, while overexpression of c-Myc or MARCKSL1 signi cantly promoted the proliferation of ACHN and OS-RC-2 cells, suggesting that overexpression of c-Myc or MARCKSL1 rescued the inhibition of circPLIN2 knockdown on the proliferation of ccRCC cells (Fig. 5A-B). Similar results were obtained in the colony formation assays. Overexpression of c-Myc or MARCKSL1 rescued the long-term inhibition of circPLIN2 knockdown on the proliferation of ccRCC cells (Fig. 5C-D). Furthermore, the results of woundhealing assays showed that knockdown of circPLIN2 drastically decreased the wound-healing speed of ACHN and OS-RC-2 cells, while overexpression of c-Myc or MARCKSL1 markedly accelerated the woundhealing speed of ACHN and OS-RC-2 cells, revealing that overexpression of c-Myc or MARCKSL1 rescued the inhibition of circPLIN2 knockdown on the migration of ccRCC cells (Fig. 5E-F). In addition, the Matrigel Transwell assays indicated that overexpression of c-Myc or MARCKSL1 signi cantly rescued the suppression of circPLIN2 knockdown on the invasion of ccRCC cells in vitro (Fig. 5G-H). Taken together, overexpression of c-Myc or MARCKSL1 rescued the inhibition of circPLIN2 knockdown on the proliferation, migration and invasion of ccRCC cells in vitro, suggesting that c-Myc and MARCKSL1 mediated the circPLIN2-regulated development and progression of ccRCC.
circPLIN2 competitively sponges miR-199a-3p to abolish the repressive effect of miR-199a-3p on ZEB1 Increasing evidence has shown that circRNAs can function as sponges for miRNAs to regulate the expression of genes by the competing endogenous RNA (ceRNA) mechanism [11][12][13][14]. Given that circPLIN2 was preferentially distributed in the cytoplasm (Fig. 2E-H), we investigated whether circPLIN2 might also function by a ceRNA mechanism. We rst made predictions through the circBank (http://www.circbank.cn/index.html) database and selected 10 miRNAs that might be sponged by circPLIN2 for further validation (Fig. 6A). The dual-luciferase reporter assays showed that miR-199a-3p had a particularly signi cant inhibitory effect on the luciferase activity of circPLIN2, suggesting that circPLIN2 might sponge miR-199a-3p (Fig. 6B). To further verify that circPLIN2 sponged miR-199a-3p, we constructed a circPLIN2 dual-luciferase reporter with the mutated miR-199a-3p binding site (Supplementary Fig. 2A). The results of dual-luciferase reporter assays showed that the wild-type (WT) circPLIN2 luciferase activity was signi cantly inhibited by miR-199a-3p, while the mutated (MUT) circPLIN2 luciferase activity was not affected (Fig. 6C). In addition, the results of RNA immunoprecipitation assays showed that circPLIN2 was drastically enriched on AGO2 protein compared with the control IgG, and the enrichment of circPLIN2 on AGO2 protein was further increased when miR-199a-3p was added (Fig. 6D). These data revealed that circPLIN2 sponged miR-199a-3p ( Fig. 6A-D).
Next, we considered whether there was a ceRNA mechanism among circPLIN2, miR-199a-3p and ZEB1. The RT-qPCR results showed that miR-199a-3p signi cantly reduced the expression level of ZEB1, while overexpression of circPLIN2 abolished the repressive effect of miR-199a-3p on ZEB1 expression (Fig. 6L-M). Additionally, the results of the dual-luciferase reporter assays indicated that overexpression of circPLIN2 signi cantly increased wild-type ZEB1 luciferase activity, while knockdown of circPLIN2 markedly decreased wild-type ZEB1 luciferase activity (Fig. 6N). Moreover, the mutated ZEB1 luciferase activity was not affected by circPLIN2 overexpression or knockdown (Fig. 6N). These results revealed that there was an endogenous RNA competition relationship between circPLIN2 and ZEB1 for miR-199a-3p. Collectively, these results suggested that circPLIN2 competitively sponged miR-199a-3p to abolish the repressive effect of miR-199a-3p on ZEB1.
circPLIN2 exerts its carcinogenic effects on ccRCC cells via the miR-199a-3p/ZEB1 axis in vitro Next, we investigated whether the circPLIN2/miR-199a-3p/ZEB1 molecular signaling pathway participated in the development and progression of ccRCC. The results of CCK-8 cell viability assays showed that knockdown of circPLIN2 signi cantly repressed the proliferation of ACHN and OS-RC-2 cells, and the proliferation of ACHN and OS-RC-2 cells was further inhibited when miR-199a-3p was added ( Fig.   7A-B). Overexpression of ZEB1 rescued the inhibition of circPLIN2 knockdown and addition of miR-199a-3p on the proliferation of ccRCC cells (Fig. 7A-B). Similar results were obtained in the colony formation assays. Overexpression of ZEB1 drastically rescued the long-term suppression of circPLIN2 knockdown and addition of miR-199a-3p on the proliferation of ccRCC cells (Fig. 7C-D). Furthermore, the woundhealing assays indicated that knockdown of circPLIN2 markedly reduced the wound-healing speeds of ACHN and OS-RC-2 cells, and the wound-healing speeds of ACHN and OS-RC-2 cells were slower when miR-199a-3p was added, while overexpression of ZEB1 signi cantly rescued the inhibition of circPLIN2 knockdown and the addition of miR-199a-3p on the migration of ccRCC cells (Fig. 7E-F). Moreover, the results of Matrigel Transwell assays showed that overexpression of ZEB1 drastically rescued the repression of circPLIN2 knockdown and the addition of miR-199a-3p on the invasion of ccRCC cells in vitro (Fig. 7G-H). Overall, our data suggested that the circPLIN2/miR-199a-3p/ZEB1 molecular signaling pathway was involved in the proliferation, migration and invasion of ccRCC cells.

circPLIN2 promotes ccRCC tumor growth in vivo
To examine the effect of circPLIN2 on the growth of ccRCC cells in vivo, we constructed subcutaneous xenograft tumors of ACHN with stable low or high expression of circPLIN2 in BALB/c nude mice. Photographs of the tumors at necropsy showed that stable knockdown of circPLIN2 signi cantly inhibited the growth of ACHN cells in vivo (Fig. 8A), while stable overexpression of circPLIN2 drastically promoted the growth of ACHN cells in vivo (Fig. 8B). In addition, the volumes of subcutaneous xenograft tumors indicated that stable knockdown of circPLIN2 markedly decreased the volumes of tumors in nude mice compared with the control group (Fig. 8C), whereas stable overexpression of circPLIN2 suggested the opposite results (Fig. 8D), which was consistent with the results of weight measurement of subcutaneous xenograft tumors (Fig. 8E-F). Collectively, these results revealed that circPLIN2 promoted the growth of ccRCC cells in vivo.

Discussion
In this study, we proved the oncogenic roles of circPLIN2 and determined its underlying mechanism in the development and progression of ccRCC. We rst explored the expression pro les of circRNAs in 10 paired samples of RCC from GSE124453 and GSE108735 in the GEO database. We initially identi ed hsa_circ_0086457, designated circPLIN2, which was derived from exons 4 to 5 of the PLIN2 gene. circPLIN2 was signi cantly upregulated in ccRCC cells and tissues, and its overexpression was correlated with higher clinical stage and worse prognosis in ccRCC patients. We identi ed the characteristics of circPLIN2 in ccRCC cells and found that circPLIN2 was preferentially distributed in the cytoplasm of ccRCC cells and had a longer half-life and a stronger resistance to digestion by RNase R treatment than its linear counterpart PLIN2. Intriguingly, depletion of circPLIN2 signi cantly attenuated the proliferation, migration and invasion of ccRCC cells in vitro and the tumor growth of ccRCC in vivo, whereas overexpression of circPLIN2 resulted in the opposite effects, suggesting that elevated circPLIN2 may be a cancer-promoting event in ccRCC. Mechanistically, circPLIN2 not only enhanced the mRNA stability of c-Myc and MARCKSL1 by binding to the KH domains of IGF2BP proteins but also competitively sponged miR-199a-3p to abolish the repressive effect of miR-199a-3p on ZEB1, which ultimately resulted in tumorigenesis and progression of ccRCC. Together, these ndings indicated the oncogenic function of circPLIN2 and its potential molecular mechanism in which elevated circPLIN2 participated in the development and progression of ccRCC by binding IGF2BP proteins and miR-199a-3p to regulate their target gene expression, including c-Myc, MARCKSL1 and ZEB1 (Fig. 8G).
Recent evidence has shown that circRNAs play vital roles in the development and progression of ccRCC [36][37][38][39]. For example, circZNF609, which is highly expressed in various ccRCC cell lines, acts as a sponge for miR-138-5p to upregulate the expression of FOXP4 and promote the growth and invasion of ccRCC [36]. Intriguingly, it has been shown that circZNF609 in myoblasts can be translated into a functional small protein [40], which is regulated by its own m 6 A modi cation [41]. Hence, we considered whether circZNF609 has a translational protein and regulatory role in ccRCC, which requires further exploration in the future. As another example showed, circTLK1 was not merely drastically upregulated in ccRCC cells and tissues but was related to the distant metastasis of tumors and the prognosis of ccRCC patients [37].
Moreover, circTLK1 upregulated the expression of CBX4 by competitively sponging miR-136-5p to exert its oncogenic activity [37]. Although these circRNAs have been shown to be involved in the development and progression of ccRCC, their key regulatory roles and molecular mechanisms have not been fully clari ed. In addition, novel circRNAs need to be further identi ed in ccRCC.
In this study, to more accurately detect the expression pro les of circRNAs in RCC, we selected a total of 10 paired RCC samples of circRNA expression data from GSE124453 and GSE108735 in the GEO database for joint analysis, which can reduce the bias of RCC sample selection in two different studies and expand the RCC sample size to make circRNA expression data more reliable. We found that circPLIN2, as an oncogene, was signi cantly highly expressed in ccRCC cells and tissues, and its overexpression was correlated with higher clinical stage and worse prognosis in ccRCC patients. Furthermore, elevated circPLIN2 promoted ccRCC cell proliferation, migration and invasion in vitro and ccRCC tumor growth in vivo. This is similar to the performance and function of circTLK1, circSDHC and circPRRC2A in ccRCC [37,42,43]. However, unlike circPLIN2, circRAPGEF5 and circAKT3 were signi cantly expressed at low levels in ccRCC and inhibited the malignant progression of ccRCC [44,45]. Overall, these con icting results surrounding circRNA performance in ccRCC can be partly explained by the fact that circRNAs participate in different molecular signaling pathways.
Notably, based on the prediction results of the circBank database, although circPLIN2 has an open reading frame (ORF), circPLIN2 lacks the internal ribosome entry site (IRES) elements [46] and m 6 A (N 6methyladenosine) modi cation [24,47] that are required for the translation of circRNAs [48]; therefore, circPLIN2 may not have translation potential. We speculate that circPLIN2 may regulate the expression of downstream genes by binding to proteins or sponging miRNAs in the cytoplasm. In this study, we demonstrated the binding of circPLIN2 and IGF2BP proteins, including IGF2BP1, IGF2BP2 and IGF2BP3 proteins. IGF2BP proteins, mainly enriched in the cytoplasm, can recognize and bind target mRNAs in an m 6 A-dependent manner and play a role as stabilizers to inhibit the degradation of their target mRNAs [35]. The IGF2BP proteins have six key domains, including the RRM1-2 domains and KH1-4 domains, and the KH1-4 domains are key for the binding of IGF2BP proteins and target mRNAs (c-Myc or MARCKSL1 mRNA) [35]. Interestingly, the speci c circPLIN2 binding domains of IGF2BP proteins happened to be the KH1-4 domains, which suggested that the KH1-4 domains might provide a common place for action among circPLIN2, IGF2BP proteins and c-Myc or MARCKSL1 mRNA. In our study, circPLIN2 enhanced the mRNA stability of c-Myc and MARCKSL1 by binding to the KH domains of IGF2BP proteins. Moreover, it has been shown that c-Myc [49,50] and MARCKSL1 [51,52], as oncogenes, participate in the development and progression of cancers. Subsequent rescue assays showed that overexpression of c-Myc or MARCKSL1 signi cantly rescued the inhibition of circPLIN2 knockdown on the proliferation, migration and invasion of ccRCC cells, suggesting that c-Myc and MARCKSL1 were involved in the circPLIN2-regulated development and progression of ccRCC.
In addition, we found an underlying ceRNA mechanism in which circPLIN2 competitively sponged miR-199a-3p to abolish the repressive effect of miR-199a-3p on ZEB1. Subsequent rescue assays further showed that the circPLIN2/miR-199a-3p/ZEB1 molecular signaling pathway participated in the development and progression of ccRCC. Intriguingly, ZEB1, as a transcriptional repressor, inhibits the transcription of E-cadherin by recruiting BRG1 and promotes epithelial-mesenchymal transition (EMT) and tumor progression [53]. Hence, we speculate that EMT may be involved in the circPLIN2-regulated development and progression of ccRCC, which needs to be further evaluated. Moreover, ZEB1 suppresses the expression of stemness-inhibiting miR-200 and miR-203 and promotes tumor proliferation and progression [54]. Therefore, we speculate that miR-200 and miR-203 may also participate in circPLIN2mediated ccRCC progression, which also needs to be further con rmed.
In conclusion, our study suggested that circPLIN2 functioned as an oncogene and participated in the development and progression of ccRCC. Additionally, our results revealed that circPLIN2 not only regulated the mRNA stability of c-Myc and MARCKSL1 by binding to the KH domains of IGF2BP proteins but also sponged miR-199a-3p to abolish the repressive effect of miR-199a-3p on ZEB1, which ultimately resulted in tumorigenesis and progression of ccRCC. These data revealed that circPLIN2 may serve as a promising diagnostic and prognostic biomarker as well as a potential therapeutic target for ccRCC patients.

Bioinformatics analysis of the expression pro le of circRNAs in RCC
We rst retrieved circRNA expression data in RCC from the GEO database (http://www.ncbi.nlm.nih.gov/geo) and obtained GSE124453 and GSE108735 data. Then, we downloaded the raw data of GSE124453 and GSE108735 from the SRA database (https://www.ncbi.nlm.nih.gov/sra) and converted them into FASTQ format using Sratoolkit software (version 2.9.2) (https://hpc.nih.gov/apps/sratoolkit.html). The FASTQ les were aligned onto the human hg38 reference using STAR software (version 2.7.1a) (https://github.com/alexdobin/STAR) [55]. circRNAs were subsequently calculated and identi ed using DCC software (https://github.com/dieterich-lab/DCC) with default parameters [56]. Next, the circRNAs identi ed were ltered by read count more than 5 and expressed samples over 30%. The function and identities of circRNAs were then annotated by the circBase database (http://www.circbase.org) [57]. DESeq2 was used to read the raw count matrix after ltration, and normalization was performed by using the variance stabilizing transformation algorithm [58]. Signi cantly differentially expressed circRNAs between RCC and normal samples were screened with the criteria of adjusted p value less than 0.05 and absolute value of log 2 (fold change) more than 2. The results of the bioinformatics analysis were eventually visualized as a heatmap and a volcano plot.

Plasmid construction and cell transfection assay
Referring to the method for constructing the circTP63 overexpression vector described in a previous study [59], we successfully constructed the circPLIN2 overexpression vector by homologous recombination using the pLCDH-ciR plasmid. For cell transfection assays, brie y, cells were rst seeded on 6-well plates to a con uence of approximately 50%. Next, cells were transfected with circPLIN2 or vector using Lipofectamine 2000 reagent according to the manufacturer's protocol and then cultured at 37 °C with 5% CO2 for 48~72 h. Finally, the expression level of circPLIN2 was assessed by RT-qPCR. Additionally, the overexpression vectors of c-Myc, MARCKSL1 and ZEB1 were designed and constructed by GENE (Shanghai, China) using the GV658 plasmid. GFP-tagged wild-type and truncated IGF2BP vectors were designed and constructed by GENE using the pEGFP-C2 plasmid. The primers used for plasmid construction are listed in Supplementary Table 4.

Statistical analysis
The IBM SPSS package (version 23.0) and GraphPad Prism software (version 6.0) were used for statistical analysis. All data in this study are shown as the means ± S.D. of the values from triplicate assays. Two-tailed Student's t test was used to compare two independent groups. Spearman's test was performed to analyze the correlations for categorical variables. The Kaplan-Meier test was performed for the univariate analysis of overall survival, and the Cox proportional hazards regression model was used for the multivariate analysis of overall survival. *p < 0.05, **p < 0.01 and ***p < 0.001 were considered statistically signi cant. 2. *, p < 0.05; **, p < 0.01; ***, p < 0.001. which is considered as a signi cant difference.  and OS-RC-2 (H) cells. All probes are labeled with Cy3. 18S was used as a cytoplasmic positive control, and U6 was used as a nuclear positive control. Two-tailed Student's t test (C). The error bars represent S.D. (n=3). ns, no signi cance; ***p < 0.001.  circPLIN2 competitively sponges miR-199a-3p to abolish the repressive effect of miR-199a-3p on ZEB1. A A sketch map was drawn to show circPLIN2 sponging 10 miRNAs predicted in the circBank database. B Dual-luciferase reporter assays for the luciferase activity of circPLIN2 in 293T cells treated with different miRNAs. Luciferase activity was normalized to re y luciferase activity. C Dual-luciferase reporter assays for the luciferase activity of circPLIN2 in 293T cells treated with pmiR-circPLIN2-WT or pmiR-circPLIN2-

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.